[Role of the immune system in SARS-CoV-2 infection: immunopathology of COVID-19]. / Papel del sistema inmune en la infección por el SARS-CoV-2: inmunopatología de la COVID-19.

The immune system is capable of adequately controlling SARS-CoV-2 infection in 81% of patients, whose disease is asymptomatic or who experience moderate symptoms. However, 19% of infected patients develop severe disease which can become critical or fatal. This review article intends to provide an overview of the epidemiological antecedents of ß-coronaviruses, describe the mechanisms of SARS-CoV-2 infection, and summarize the rational immunological underpinnings known at present which allow for a better understanding of the immunopathology of COVID-19. The SARS-CoV-2 virus is capable of profoundly altering the behavior of molecular and cellular components of the immune system. The initial decisions of the innate immune system are responsible for a proper or improper response of the adaptive immune system and, along with comorbidities, are directly associated with disease progression.

Colecistectomía y nefrectomía radical por puerto único umbilical en un mismo acto quirúrgico / Trans-umbilical single-port radical nephrectomy with concomitant cholecystectomy

Objetivos: La evolución de los procedimientos laparoscópicos hace que sea necesario en ocasiones, la realización de cirugías combinadas con otras especialidades. Actualmente, algunas de estas intervenciones se pueden realizar mediante cirugía por puerto único umbilical. Material y métodos: Presentamos el caso de un paciente con tumoración renal de 4,5 cm y colelitiasis al que realizamos colecistectomía y nefrectomía radical derecha por puerto único umbilical de manera concomitante. Este es el primer caso publicado en España en el que se realiza esta intervención combinada mediante cirugía de puerto único umbilical. Resultados: Se realizó la cirugía sin complicaciones intraoperatorias ni postoperatorias, con un correcto control de la patología tumoral y un excelente resultado estético. Conclusión: En las situaciones que sean necesarias realizar un abordaje multidisciplinar con otras especialidades quirúrgicas involucradas, estas se pueden realizar mediante cirugía por puerto único umbilical, con las ventajas de una menor morbilidad postoperatoria y un mejor resultado estético

Objectives: Occasionally, the development of laparoscopic procedures allows the performance of combined surgeries. Currently, some of these can be carried out by trans-umbilical single-port laparoscopy. Material and methods: We report a patient with renal tumor of 4.5 cm and cholelithiasis who undergone to trans-umbilical single port-right radical nephrectomy with concomitant cholecystectomy. This is the first case reported in Spain that this combined procedure is performed using umbilical single port surgery. Results: No complications (intra or postoperative) have been described in this case, achieving proper control of tumor pathology and an excellent cosmetic outcome. Conclusion: In those cases in which multidisciplinary approach is required, surgery can be performed by trans-umbilical single-port laparoscopy as consequence of its reduced postoperative morbidity and better cosmetic results

Trans-umbilical single-port radical nephrectomy with concomitant cholecystectomy.

OBJECTIVES: Occasionally, the development of laparoscopic procedures allows the performance of combined surgeries. Currently, some of these can be carried out by trans-umbilical single-port laparoscopy. MATERIAL AND METHODS: We report a patient with renal tumor of 4.5cm and cholelithiasis who undergone to trans-umbilical single port-right radical nephrectomy with concomitant cholecystectomy. This is the first case reported in Spain that this combined procedure is performed using umbilical single port surgery. RESULTS: No complications (intra or postoperative) have been described in this case, achieving proper control of tumor pathology and an excellent cosmetic outcome. CONCLUSION: In those cases in which multidisciplinary approach is required, surgery can be performed by trans-umbilical single-port laparoscopy as consequence of its reduced postoperative morbidity and better cosmetic results.

A tumor suppressor role for srGAP3 in mammary epithelial cells.

srGAP3, a member of the Slit-Robo sub-family of Rho GTPase-activating proteins (Rho GAPs), controls actin and microtubule dynamics through negative regulation of Rac. Here, we describe a potential role for srGAP3 as a tumor suppressor in mammary epithelial cells. We show that RNAi-mediated depletion of srGAP3 promotes Rac dependent, anchorage-independent growth of partially transformed human mammary epithelial cells (HMECs). Furthermore, srGAP3 expression is absent, or significantly reduced in 7/10 breast cancer cell lines compared with normal HMECs. Re-expression of srGAP3 in a subset of these cell lines inhibits both anchorage-independent growth and cell invasion in a GAP-dependent manner, and this is accompanied by an increase in phosphorylation of the ezrin/radixin/moesin (ERM) family proteins and myosin light chain 2 (MLC2). Inhibition of the Rho regulated kinase, ROCK, reduces ERM and MLC2 phosphorylation and restores invasion. We conclude that srGAP3 has tumor suppressor-like activity in HMECs, likely through its activity as a negative regulator of Rac1.

Mortalidad infantil en un hospital de nivel terciario. Limitación de esfuerzo terapéutico, correspondencia clínico-patológica y precisión diagnóstica / Infant mortality in a third level paediatric hospital. therapeutic effort limitation, clinical-pathological agreement and diagnostic accuracy

Objetivo: Analizar la mortalidad en un hospital infantil de tercer nivel y alta complejidad. Material y métodos: Se revisaron los fallecidos en el Hospital Infantil La Paz durante los años 2007, 2008 y 2009. Se analizaron datos epidemiológicos, diagnósticos clínicos y de autopsia y su correspondencia, y si se llegaba a un diagnóstico etiológico definitivo. La limitación del esfuerzo terapéutico y la previsibilidad del fallecimiento también fueron recogidas. Las variables fueron prospectivamente definidas al inicio. Resultados: Se estudiaron 253 fallecimientos (6,08 por mil ingresos). El 43,4% eran menores de 1 mes y el 63,9% menores de un año. La patología neonatal y la hemato-oncológica fueron las causas más frecuentes. Fallecieron en las tres unidades de cuidado intensivo el 87%. Se practicó autopsia a 53% de los fallecidos y se detectó un 7,8% de nuevos hallazgos significativos, aunque solo en un caso podría el tratamiento haber modificado el pronóstico. Limitación de esfuerzo terapéutico y cuidado paliativo se instauró en el 41,9%. El fallecimiento era esperado al inicio del proceso en 83,9%, En 92% se consideró que existía un diagnóstico definitivo y en 86,4% un diagnóstico etiológico de los procesos que condujeron al fallecimiento. Conclusiones: El análisis de la mortalidad hospitalaria permite evaluar la calidad de la asistencia pediátrica y detectar resultados adversos. La autopsia continúa proporcionando información relevante. La limitación de esfuerzo terapéutico y cuidado paliativo es una medida cada vez más frecuente en la edad pediátrica. El número de niños que muere sin diagnóstico etiológico sigue siendo alto(AU)

Objective: To study infant and child mortality in a third level children's hospital treating highly complex patients. Patients and methods: All children dying in the period 2007- 2009 at La Paz Children's Hospital were evaluated. Epidemiological data, autopsy rate, clinical and autopsy diagnoses and their correspondence and the number of, patients with precise final diagnoses were analysed. Therapeutic effort limitation and palliative care were also evaluated as well as if the final result was expected according to the initial disease or clinical condition of the patients. All the variables were prospectively defined at the start of the study period. Results: A total of 253 cases (6.08‰ admissions) were analysed. The two leading causes of death were disorders related to prematurity and low birth weight, and haematology oncology malignant diseases. Most patients (87%) died in an intensive care unit (neonatal or paediatric). During the study period 134 autopsies (53%) were performed, and new clinically significant findings were observed in 12 of these (7.8%) but in only one case the treatment could have possibly modified the prognosis (class I discrepancy). Therapeutic effort limitation and palliative care were implemented in 41.9%. Death was initially expected in 83.9% of cases. An accurate final diagnosis was defined in 92%, and the aetiology of the disease was considered to be identified in 86.4% of all deaths. Conclusions: Hospital mortality analysis is useful to evaluate the quality of the paediatric care and to detect adverse results that could be corrected. Paediatric autopsy continues to provide clinically significant data for paediatricians and families. Therapeutic effort limitation and palliative care is increasingly applied in paediatric end of life care. The number of infants and children dying without a final aetiological diagnosis is still considerably high(AU)

[Infant mortality in a third level paediatric hospital. therapeutic effort limitation, clinical-pathological agreement and diagnostic accuracy]. / Mortalidad infantil en un hospital de nivel terciario. Limitación de esfuerzo terapéutico, correspondencia clínico-patológica y precisión diagnóstica.

OBJECTIVE: To study infant and child mortality in a third level children's hospital treating highly complex patients. PATIENTS AND METHODS: All children dying in the period 2007- 2009 at La Paz Children's Hospital were evaluated. Epidemiological data, autopsy rate, clinical and autopsy diagnoses and their correspondence and the number of, patients with precise final diagnoses were analysed. Therapeutic effort limitation and palliative care were also evaluated as well as if the final result was expected according to the initial disease or clinical condition of the patients. All the variables were prospectively defined at the start of the study period. RESULTS: A total of 253 cases (6.08‰ admissions) were analysed. The two leading causes of death were disorders related to prematurity and low birth weight, and haematology oncology malignant diseases. Most patients (87%) died in an intensive care unit (neonatal or paediatric). During the study period 134 autopsies (53%) were performed, and new clinically significant findings were observed in 12 of these (7.8%) but in only one case the treatment could have possibly modified the prognosis (class I discrepancy). Therapeutic effort limitation and palliative care were implemented in 41.9%. Death was initially expected in 83.9% of cases. An accurate final diagnosis was defined in 92%, and the aetiology of the disease was considered to be identified in 86.4% of all deaths. CONCLUSIONS: Hospital mortality analysis is useful to evaluate the quality of the paediatric care and to detect adverse results that could be corrected. Paediatric autopsy continues to provide clinically significant data for paediatricians and families. Therapeutic effort limitation and palliative care is increasingly applied in paediatric end of life care. The number of infants and children dying without a final aetiological diagnosis is still considerably high.

Exploring mass spectrometry suitability to examine human liver graft metabonomic profiles.

BACKGROUND: Transplant surgeons rely on morphologic aspects of the organ as well as clinical and histologic data to decide whether to use a graft. Metabonomics measures the "downstream" products of proteins and genes; these metabolic profiles are particularly good reporters of tissue physiologic features. Sample preparation and data acquisition are generally considered limiting steps in metabonome analysis because they are important sources of variability. State-of-the-art mass spectrometry and multivariate statistical analysis have been used to explore the suitability of a metabonomic platform as a liver tissue metabonomic profiling method. OBJECTIVE: To develop robust and reliable sample processing and mass spectrometry protocols for studying human liver metabonomic profiles. MATERIALS AND METHODS: Liquid chromatography coupled with mass spectrometry was used to analyze 20 liver tissue samples from 10 discarded and 10 transplanted grafts. Principal component analysis (PCA) and projection to latent structures-discriminant analysis (PLS-DA) were used for data interpretation. RESULTS: Standard operating protocols for sample processing (tissue homogenization) and data acquisition were developed. The quantification of the quality controls present in the test mix demonstrated coefficients of variation less than 15%. The PCA score plot revealed that the sample triplicate cluster was quite close. Furthermore, PLS-DA analysis demonstrated a clear separation (transplanted vs discarded) along the first component. DISCUSSION: Multivariate data analysis (PCA and PLS-DA) indicated that protocols developed in-house for sample processing and mass spectrometry data acquisition were sufficiently sensitive (approximately 1245 features) and reproducible (sample triplicate clusters and test mix quantification) to perform liver tissue metabonomic profiling. In addition, a reduced set of metabolites was selected as potential biomarkers responsible for sample discrimination. These findings encourage ongoing research into the development of a metabonomic model to assess liver graft quality and function before transplantation.

In vitro evaluation of potential hepatotoxicity induced by drugs.

The liver is the most important target for toxicity caused by drugs. This vulnerability is a consequence of the functional features of the liver and their role in the metabolic elimination of most drugs. Therefore, evaluation of potential hepatotoxicity represents a critical step in the development of new drugs. The liver is very active in metabolising foreign compounds and, although biotransformation reactions generally parallel detoxification processes, the formation of reactive metabolites is relatively frequent. Thus, drug-induced hepatotoxicity can be due to the administered compound itself or to metabolites formed by hepatic metabolism. The most important systems to study hepatotoxicity and metabolic activity in vitro are liver slices, isolated liver cells in suspensions or in primary cultures including co-culture methods and special 3D techniques, various subcellular fractions and hepatic cell lines. These models can be used for cytotoxicity and genotoxicity screening, and also to identify the mechanisms involved in drug-induced hepatotoxicity. Assessment of current cytotoxicity and hepatic-specific biochemical effects are limited by the inability to measure a wide spectrum of potential mechanistic changes involved in the drug-induced toxic injury. A convenient selection of end-points allows a multiparametric evaluation of drug toxicity. In this regard, omic (cytomic, metabonomic, proteomic and toxicogemic) approaches help defining patterns of hepatotoxicity for early identification of potential adverse effects of the drug to the liver. The development of robust in vitro-based multiparametric screening assays covering a wider spectrum of key effects will heighten the predictive capacity for human hepatotoxicity, and accelerate the drug development process.

Functional characterization of hepatocytes for cell transplantation: customized cell preparation for each receptor.

The first indication of hepatocyte transplantation is inborn liver-based metabolic disorders. Among these, urea cycle disorders leading to the impairment to detoxify ammonia and Crigler-Najjar Syndrome type I, a deficiency in the hepatic UDP-glucuronosyltransferase 1A1 present the highest incidence. Metabolically qualified human hepatocytes are required for clinical infusion. We proposed fast and sensitive procedures to determine their suitability for transplantation. For this purpose, viability, attachment efficiency, and metabolic functionality (ureogenic capability, cytochrome P450, and phase II activities) are assayed prior to clinical cell infusion to determine the quality of hepatocytes. Moreover, the evaluation of urea synthesis from ammonia and UDP-glucuronosyltransferase 1A1 activity, a newly developed assay using beta-estradiol as substrate, allows the possibility of customizing cell preparation for receptors with urea cycle disorders or Crigler-Najjar Syndrome type I. Sources of human liver and factors derived from the procurement of the liver sample (warm and cold ischemia) have also been investigated. The results show that grafts with a cold ischemia time exceeding 15 h and steatosis should not be accepted for hepatocyte transplantation. Finally, livers from non-heart-beating donors are apparently a potential suitable source of hepatocytes, which could enlarge the liver donor pool.

Sequential hepatogenic transdifferentiation of adipose tissue-derived stem cells: relevance of different extracellular signaling molecules, transcription factors involved, and expression of new key marker genes.

Adipose tissue contains a mesenchymal stem cell (MSC) population known as adipose-derived stem cells (ASCs) capable of differentiating into different cell types. Our aim was to induce hepatic transdifferentiation of ASCs by sequential exposure to several combinations of cytokines, growth factors, and hormones. The most efficient hepatogenic protocol includes fibroblastic growth factors (FGF) 2 and 4 and epidermal growth factor (EGF) (step 1), hepatocyte growth factor (HGF), FGF2, FGF4, and nicotinamide (Nic) (step 2), and oncostatin M (OSM), dexamethasone (Dex), and insulin-tranferrin-selenium (step 3). This protocol activated transcription factors [GATA6, Hex, CCAAT/enhancer binding protein alpha and beta (CEBPalpha and beta), peroxisome proliferator-activated receptor-gamma, coactivator 1 alpha (PGC1alpha), and hepatocyte nuclear factor 4 alpha (HNF4alpha)], which promoted a characteristic hepatic phenotype, as assessed by new informative markers for the step-by-step hepatic transdifferentiation of hMSC [early markers: albumin (ALB), alpha-2-macroglobuline (alpha2M), complement protein C3 (C3), and selenoprotein P1 (SEPP1); late markers: cytochrome P450 3A4 (CYP3A4), apolipoprotein E (APOE), acyl-CoA synthetase long-chain family member 1 (ACSL1), and angiotensin II receptor, type 1 (AGTR1)]. The loss of adipose adult stem cell phenotype was detected by losing expression of Thy1 and inhibitor of DNA binding 3 (Id3). The reexpression of phosphoenolpyruvate corboxykinase (PEPCK), apolipoprotein C3 (APOCIII), aldolase B (ALDOB), and cytochrome P450 1A2 (CYP1A2) was achieved by transduction with a recombinant adenovirus for HNF4alpha and finally hepatic functionality was also assessed by analyzing specific biochemical markers. We conclude that ASCs could represent an alternative tool in clinical therapy for liver dysfunction and regenerative medicine.

Desarrollo, análisis y optimización de modelos celulares hepáticos para estudios de fármacotoxicología y terapia celular / Development and optimization of hepatic cell models for pharmaco-toxicology and cell therapy research

El hígado juega un papel fundamental en el metabolismo de medicamentos y enel mantenimiento de la homeostasis del organismo y, por tanto los modelos celulareshepáticos desempeñan un papel clave para estudios fármaco-toxicológicos ymás recientemente en el campo de la terapia celular. Sin embargo, la limitada disponibilidadde hepatocitos viables y funcionales, debido a la falta de tejido hepático,es la principal limitación para utilizar estos recursos celulares. El objetivo delpresente trabajo se ha basado en el desarrollo y caracterización de modelos celulareshepáticos que puedan constituir una alternativa a los hepatocitos para estetipo de aplicaciones. Para ello se han abordado tres estrategias diferentes: 1) optimizacióndel proceso de obtención de hepatocitos a partir de hígados enterosdescartados para transplante, determinando las condiciones adecuadas para elaislamiento y cultivo de hepatocitos; 2) caracterización funcional de las células delhepatoblastoma HepG2, y 3) desarrollo de un protocolo para inducir la diferenciaciónhepatogénica de células madre mesenquimales adultas derivadas de tejidoadiposo (ADSC). Para conseguir un buen aprovechamiento de hígados descartadospara transplante resulta necesario optimizar los protocolos de aislamiento y criopreservaciónde hepatocitos. El estudio con células madre adultas se presenta comouna alternativa muy válida para la obtención de hepatocitos-like viables y funcionalmenteactivos, útiles a corto plazo en estudios de fármaco-toxicología y en unfuturo para terapia celular hepática. El uso de células madre abre un gran abanicode posibilidades, facilitando el establecimiento de un modelo celular diferenciadoadulto con características que otros modelos celulares, como son el hepatomahumano HepG2, no presentan. No obstante, es necesario adquirir un mayor conocimientode los mecanismos celulares y moleculares que controlan la transdiferenciacióna hepatocitos

Given the importance of the liver in the metabolism and maintenance of thehomeostasis of the organism, many studies have been conducted in the area oftoxicology and, more recently, in hepatic cellular therapy. However, the maindrawback is the limited availability of viable and functional hepatocytes due tothe scarcity of liver tissue. The purpose of this work was based on the developmentand characterization of hepatic cellular models to become an alternative tohepatocytes in toxicology studies and cellular therapy. To this end, three mainobjectives have been investigated: 1) to adopt a procedure of hepatocyte isolationfrom discarded organs for transplantation which determines the optimal conditions for the isolation and culture of hepatocytes, 2) to characterize the cells from thehepatoblastome HepG2, and 3) to develop a hepatogenic differentiation protocolto induce the hepatic differentiation in adipose-derived stem cells (ADSC). Inparticular, the hepatogenic differentiation of stem cells opens a wide range ofpossibilities to facilitate the establishment of an adult differentiated cellular modeluseful for pharmaco-toxicological studies and for hepatic cellular therapy. The useof adult stem cells may allow the establishment of an adult cellular model withproperties that others cellular models, like HepG2, do not show. However, it isnecessary to optimise the isolation and cryopreservation procedures, as well as thedifferentiation protocols from adult stem cells and try to acquire a wide knowledgeof the cellular and molecular mechanisms that control the transdifferentiation tohepatocytes

Interactions of polyphenols with the P450 system: possible implications on human therapeutics.

Polyphenols are a family of natural compounds with many biological properties. This review focuses on their potential interaction on the cytochrome P450 system. Effects of phenolic acids, anthocyanins, stilbenes, catechins and other flavonoids on the drug metabolising function are revised. Their daily intake and presence in herbal medicines justify the study of potential drug-interaction to prevent undesirable clinical consequences.

Cell lines: a tool for in vitro drug metabolism studies.

Primary cultured hepatocytes are a valuable in vitro model for drug metabolism studies. However, their widespread use is greatly hindered by the scarcity of suitable human liver samples. Moreover, the well-known in vitro phenotypic instability of hepatocytes, the irregular availability of fresh human liver for cell harvesting purposes, and the high batch-to-batch functional variability of hepatocyte preparations obtained from different human liver donors, seriously complicate their use in routine testing. To overcome these limitations, different cell line models have been proposed for drug metabolism screening. Human liver-derived cell lines would be ideal models for this purpose given their availability, unlimited life-span, stable phenotype, and the fact that they are easy to handle. However, the human hepatoma cells currently used (i.e. HepG2, Mz-Hep-1) show negligible levels of drug-metabolizing and do not constitute a real alternative to primary hepatocytes. Different strategies have been proposed to generate metabolically competent immortalized hepatocytes (transformation of human hepatocytes with plasmids encoding immortalizing genes, hepatocyte-like cells derived from stem cells, cell lines generated from transgenic animals, hepatocyte/hepatoma hydrid cells). Moreover, recombinant models heterologously expressing P450 enzymes in different host cells have been developed and successfully used in drug metabolism testing. In addition, new strategies have recently been explored to upregulate the expression of drug-metabolizing enzymes in cell lines of a human origin (i.e. transfection with expression vectors encoding key hepatic transcription factors). Among metabolic-based drug-drug interactions, P450 inhibition seems to be the most important. A major application of recombinant models expressing a single P450 is the screening of potential enzyme inhibitors. Therefore, pharmaceutical companies increasingly make use of cell lines to speed up the selection of new drugs with favourable pharmacokinetic and metabolic properties.

Strategies to in vitro assessment of major human CYP enzyme activities by using liquid chromatography tandem mass spectrometry.

At the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single candidate can be identified for development. Determining the role of CYP enzymes in the metabolism of a compound and evaluating the effect of NCEs on human CYP activities are key issues in pharmaceutical development as they may explain inter-subject variability, drug-drug interactions, non-linear pharmacokinetics and toxic effects. Reliable methods for determining enzyme activities are needed to characterize an individual CYP enzyme and to obtain a tool for the evaluation of its role in drug metabolism in humans. Different liquid chromatography tandem mass spectrometry methodologies have been developed for the fast and routine analysis of major in vivo and in vitro CYPs enzyme activities. The high sensitivity and selectivity of mass spectrometry allow traditional assays to be minimized, thus saving time, efforts and money. Therefore this technology has become the method of choice for the fast assessment of CYP enzyme activities in early drug discovery development. Our intention herein is to review the most recent approaches that have been developed to quickly assess CYPs activities using in vitro models and liquid chromatography coupled with mass spectrometry, as well as their application in early drug discovery.

Modulation of P450 enzymes by Cuban natural products rich in polyphenolic compounds in rat hepatocytes.

This paper reports cytotoxic effects and changes in the P450 system after exposing rat hepatocytes to four polyphenol-rich products widely used in Cuban traditional medicine (Mangifera indica L. (MSBE), Thalassia testudinum (Tt), Erythroxylum minutifolium and confusum extracts). Effects of mangiferin, the main polyphenol in MSBE, were also evaluated. Cytotoxicity was assayed by the MTT test after exposure of cells to the products (50-1000 microg/mL) for 24 or 72 h. The results showed that 500 microg/mL MSBE was moderately cytotoxic after 72 h, while mangiferin was not. Marked reductions in cell viability were produced by Erythroxylum extracts at concentrations > or = 200 microg/mL, whereas only moderate effects were induced by 1000 microg/mL Tt. Seven specific P450 activities were evaluated after 48 h exposure of cells to the products. MSBE reduced phenacetin O-deethylation (POD; CYP1A2) activity in a concentration-dependent manner (IC(50)=190 microg/mL). No decreases were observed in other activities. In contrast, mangiferin produced reductions in five P450 activities: IC(50) values of 132, 194, >200, 151 and 137 microg/ml for POD (CYP1A2), midazolam 1'-hydroxylation (M1OH; CYP3A1), diclofenac 4'-hydroxylation (D4OH; CYP2C6), S-mephenytoin 4'-hydroxylation (SM4OH), and chlorzoxazone 6-hydroxyaltion (C6OH; CYP2E1), respectively. E. minutifolium, E. confusum and Tt extracts produced small reductions in SM4OH and C6OH activities, but no significant changes were noted in the other P450 activities. On the other hand, all the products increased the benzyloxyresorufin O-debenzylation (BROD; CYP2B1) activity, with MSBE, mangiferin or E. minutifolium showing the highest effects (about 2-fold over control). Our results showed in vitro effects of these natural products on P450 systems, possibly leading to potential metabolic-based interactions.

Determination of major human cytochrome P450s activities in 96-well plates using liquid chromatography tandem mass spectrometry.

At the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single candidate can be identified for development. Evaluation of the effect of NCEs on human CYP450 enzyme activities is a key issue in pharmaceutical development as it may explain inter-subject variability, drug-drug interactions, non-linear pharmacokinetics and toxic effects. A liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method has been developed for the fast and routine analysis of major human CYP450s enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in primary hepatocyte cell cultures. The high sensitivity and selectivity of mass spectrometry has allowed traditional assays to be minimized, thus enabling the use of 96-well plate format which markedly reduced the number of hepatocytes needed for each cytochrome CYP450 activity measurement, a fact that is particularly critical concerning human hepatocytes.

Branched-chain amino acids and arginine supplementation attenuates skeletal muscle proteolysis induced by moderate exercise in young individuals.

This study aimed at evaluating the effect of a single oral intake of branched-chain amino acids (BCAA) with Arg on skeletal muscle protein metabolism during moderate exercise in young individuals. Eight healthy volunteers (4 males and 4 females, means +/- SEM, 26 +/- 1 yrs, 177.8 +/- 3.7 cm, 72.6 +/- 3.9 kg) were studied in a randomized double-blind placebo-controlled cross-over trial. The subjects performed 3 bouts of 20-min cycling exercise (5-min break between each bout) at 126 +/- 13 W corresponding to 50 % of the maximal work intensity. A single oral supplement of either a BCAA drink containing 2 g of BCAA and 0.5 g of Arg or an isocaloric placebo drink was given at 10 min of the 1st exercise bout. Both arterial and venous blood samples were simultaneously taken from the radial artery and the femoral vein, respectively. Blood flow in the femoral artery was determined using the ultrasound Doppler technique. The blood sampling and blood flow measurements were performed at rest, every 10 min during each exercise bout. Net balance of BCAA and Phe across the leg muscles were measured by the arteriovenous difference method. The BCAA ingestion resulted in increases in both the plasma BCAA concentration and BCAA uptake into the working leg. The Phe release from the leg during exercise significantly increased as compared to the basal level in the placebo trial (0.97 +/- 0.28 vs. 0.23 +/- 0.22 micromol/min, p < 0.05). In the BCAA trial, the cumulative Phe release from the leg during the 3rd exercise bout was significantly lower than that in the placebo trial (5.0 +/- 7.4 vs. 35.9 +/- 13.2 micromol/25 min, p < 0.05). These results suggest that endurance exercise at moderate intensity enhances proteolysis in working muscles, and a single oral intake of 2 g of BCAA with Arg at onset of exercise effectively suppresses exercise-induced skeletal muscle proteolysis.

In vitro ADME medium/high-throughput screening in drug preclinical development.

The study of the ADME features of the huge number of new chemical entities (NCEs) produced mainly by combinatorial chemistry has become a bottleneck in the drug development process. In response the pharmaceutical industry is involved in the development of new medium/high-throughput screening capabilities. The aim of this paper is to review some of the available in vitro ADME systems adapted to screening requirements together with the technological approaches which can be linked to medium/high-throughput molecular screening.

Remifentanilo en bolos para inducir apnea y bradicardia necesarias para la realización de coronariografía-tcmd en un paciente pediátrico / Remifentanil: bolus doses to induce apnea and bradycardia for coronary arteriography in a pediatric patient

No disponible

No disponible